The evaluation of clotting times in the laboratory detection of lupus anticoagulants.

Although there is international consensus regarding the general principles of testing for lupus anticoagulants (LAs), no agreement exists as far as the analysis of the clotting time results is concerned. Twenty-nine laboratories participating in the Fifth International Survey of Lupus Anticoagulants (ISLA-5) reported the activated partial thromboplastin time (APTT)-based clotting times obtained on seven defined test samples and a normal plasma (NP) using the same two reagents with low and high phospholipid (PL) concentrations, respectively. These clotting times were used to analyse how various methods of calculating the results may influence the apparent sensitivity of LA tests. We found that the use of a separate screening test may lead to the exclusion of samples where the presence of LA would have been detected by a combined screening and confirmatory method. For instance, the dilute APTT (dAPTT) gave a sensitivity of 53.5% (screening test), while the calculation of a ratio between the clotting times obtained with two different PL concentrations gave a sensitivity of 68.1% (confirmatory test). The normalisation of results by dividing with the corresponding results of NP increased the apparent sensitivity. The screening test ratio between dAPTT results of test samples and NP gave a sensitivity of 84.7%. The normalised ratio between the clotting times obtained with the two reagents (lupus ratio, LR) gave a sensitivity of 95.1%. We conclude that when testing for LA, all samples should be tested with both low (screening procedure) and high (confirmatory procedure) PL concentrations. These two clotting times should be evaluated in relation to each other and to the corresponding results obtained with a reference plasma (normalisation).

The Lupus Ratio test--an interlaboratory study on the detection of Lupus anticoagulants by an APTT-based, integrated, and semi-quantitative test. Fifth International Survey of Lupus Anticoagulants--ISLA 5.

The Lupus Ratio (LR) test for lupus anticoagulants integrates screening, mixing with normal plasma and confirmation procedures into one assay. The sensitivity and reproducibility of the APTT based version of this assay was tested in an interlaboratory study that was part of the Fifth International Survey of Lupus Anticoagulants (ISLA-5). One LA negative plasma containing heparin, six LA positive plasmas and a normal pooled plasma (NP) were distributed to 31 laboratories world-wide together with two APTT reagents, one with a high and one with a low phospholipid concentration. The laboratories performed two APTTs, one with each reagent, on 1:1 mixtures of test plasma and NP. The ratio between the two clotting times was divided by the corresponding ratio for the NP. This final ratio is the LR of that plasma. The overall sensitivity was found to be 95.1%, and the normal, heparin-containing sample was reported to be negative by all the laboratories. When the results were grouped in low, medium and high positive plasmas, a "consensus" regarding the strength of each plasma was easily found. 85.0% of the results were in agreement with this consensus. This study shows that with the LR test, it is possible to obtain high interlaboratory agreement regarding the presence or absence of LA as well as the semi-quantification of this inhibitor.

Lupus anticoagulants and thrombosis: clinical association of different coagulation and immunologic tests.

The dilute Russell's viper venom time (dRVVT) and the kaolin clotting time (KCT) are two among the most commonly used coagulation tests for the detection of lupus anticoagulants. The dRVVT seems superior to the KCT in identifying LA-positive patients at risk of thrombosis. However, this relationship is greatly influenced by both the source of reagents and the instrumentation employed to carry out the assays. Therefore, 4 dRVVTs ("home-made" dRVVT, DVV test, Bioclot LA, LA Screen), and one KCT (Kaoclot) were performed in two centers and compared for their retrospective correlation with the thrombotic complications of 72 patients with a previously established diagnosis of lupus anticoagulants. Two other assays ("home-made" KCT, and Colloidal Silica Clotting Time, CSCT) were performed in one of the two centers, and compared with Kaoclot for their clinical correlations in the same population of patients, 44 of whom (61%) had suffered from arterial and/or venous thrombosis. A rather good degree of inter-laboratory and inter-assay correlations of the different tests was found. However, a statistically significant association with thrombosis was found only with the coagulation profile generated using the "home-made" dRVVT. When the commercially available dRVVTs were used, none of the coagulation profiles remained associated with thrombosis. When the assays were analyzed separately, the association with thrombosis was statistically significant for LA screen (p = 0.0019), DVV test (p = 0.0043), and Bioclot (p = 0.0255), and of borderline significance for the "home-made" dRVVT (p = 0.0503) in one center. This last assay was also significantly associated with thrombosis in the other center (p = 0.0139). When venous and arterial thrombosis were considered separately, DVV test was statistically associated with venous thrombosis in both centers (p = 0.0076 and p = 0.0187, respectively), and LA screen in one center (p = 0.0303). No dRVVT was found to correlate with arterial thrombosis. Kaoclot, Colloidal Silica Clotting Time, and the "home-made" KCT did not correlate with thrombosis. The prevalence of IgG and/or IgM antibodies to cardiolipin, beta2-glycoprotein I and prothrombin were 74%, 86% and 85%, respectively. Increased titers of IgG anticardiolipin antibodies were associated with arterial thrombosis (p = 0.0375), whereas IgM anti-beta2-glycoprotein I antibodies were associated with venous thrombosis (p = 0.0433). In conclusion, these retrospective data support the notion that the dRVVT, rather than other coagulation or ELISA tests, are able to identify lupus anticoagulant-positive patients at risk of thrombosis. This property appears common to several commercially available dRVVT kits, making this type of assay the ideal target of future efforts of laboratory standardization.